The increasing spread of β-lactamase-producing pathogens represents an emerging serious public health threat specially to treat nosocomial infections. This study was carried out to determine the prevalence and molecular characterization of β-lactamase-producing Enterobacter cloacae isolates; and to estimate the prevalence of integrons in these isolates. A total of 41 clinical isolates of E. cloacae were recovered from different hospitals in the North West Bank-Palestine. Enterobacter cloacae isolates were identified using API 20E system and β-lactamase genes (ESBL, MBL and AmpC β-lactamase genes) detection was carried out using multiplex PCR technique. Results of the current research showed that the prevalence of β-lactamases among the studied clinical E. cloacae isolates was (34/41) 82.9%. The prevalence of ESBLs, MBLs and AmpC β-lactamase genes was 80.5%, 14.6% and 9.8%, respectively. For ESBL, blaTEM gene was the most dominant with a prevalence rate 63.4%. Other detected genes were 31.7%, 29.3, and 7.3% for blaOXA, blaSHV and blaCTX-M, respectively. Coexistence of 2 ESBL genes or more was detected in 39% of E. cloacae isolates. For AmpC β-lactamases only blaDHA gene was detected with a prevalence 4.9%, whereas for MBLs, the prevalence of blaIMP alone was 9.8%, blaSPM and blaIMP, and blaSIM and blaSPM together was 2.4% for each. A total of 8 isolates (19.5%) showed coexistence with at least another type of β-lactamases. In this study, class 1 integrons were detected only in β-lactamase-producing E. cloacae isolates with prevalence of (17/34) 50% among β-lactamase producers. ERIC-PCR typing of 34 clinical isolates of E. cloacae harbored different β-lactamase genes, were grouped into 5 ERIC PCR profiles (clusters) at a 70% similarity level. Results of ERIC-PCR typing showed that at lease there are 3 identical clones circulating among these hospitals and the predominant clone is C1CL1. The emergence and increase of β-lactamase-producing E. cloacae infections is worrisome. Effective measures should be taken to control the spread β-lactamase-producing bacteria.